Vaccine formulation to protect against pertussis

ABSTRACT

A vaccine composition for intranasal administration includes a Bordetella pertussis antigen, and an effective adjuvant amount of a high molecular weight glucose polymer. The high molecular weight glucose polymer may be a beta-glucan. The Bordetella pertussis antigen may be an extracellular toxin, an adhesion protein, an outer membrane protein, a receptor protein, a fragment thereof, or a mixture thereof.

TECHNICAL FIELD

Various embodiments disclosed herein relate generally to a vaccinecomposition containing Bordetella pertussis (Bp) antigens and a highmolecular weight polymer of glucose, more specifically for intranasaladministration.

BACKGROUND

Pertussis is a bacterial, airborne disease that can be spread throughcoughing and sneezing. The Gram-negative bacteria invade the respiratoryspace, propagate, and releases bacterial toxins, causing pulmonary, andif untreated, cardiac dysfunction. Commonly known as whooping cough,pertussis can be deadly to young children or immune-compromisedindividuals. Currently, the standard vaccine for pertussis, is anacellular vaccine (aP; DTaP; Tdap), which presents several proteins fromthe B. pertussis pathogen to train the human immune system to respondwith a humoral antibody response to clear the pathogen.

Despite high vaccine coverage, whooping cough has re-emerged as a majorpublic health concern in the U.S. and the world. The incidence ofpertussis has recently reached levels not seen since the 1950's. It isarguable that this increase is due to the switch from a whole cellvaccine (wP) to the currently used acellular vaccines. Originallyformulated in the 1930-40's, the whole cell bacterial vaccine reducedthe incidence of pertussis contraction but was associated with negativeside effects. To remediate this, an acellular form of the vaccine wasdeveloped in the 1980's, which contained 2-5 proteins of B. pertussisbacteria adsorbed to alum adjuvant.

The acellular vaccines were developed to direct the immune responseagainst the key components of the pathogen: 1) the extracellularpertussis toxin (PT), 2) the adhesion proteins (filamentoushemagglutinin (FHA) and fimbriae (FIM)), and 3) pertactin (PRN; anouter-membrane protein). aPs use aluminum hydroxide as the adjuvant toadsorb the antigens leading to a Th2 humoral response. In contrast,whole cell vaccines promote a Th1/Th17 response that activates both anIgG2a humoral response and cell mediated killing by macrophages andneutrophils. Natural immunity (due to infection) and wP immunizationinduced immunity lasted decades in humans.

Although acellular vaccines provide a safer alternative to whole cellvaccines, it appears that the acellular form has a shortened period ofprotection, resulting in a decreased efficacy in the years afterimmunization. This has led to a rise in the number of older children,and adolescents contracting whooping cough.

There are several hypotheses as to why whooping cough has re-emerged atsuch alarming rates. Data from the baboon model of pertussis hasindicated that while aPs protect against the disease manifestation, theaPs do not prevent colonization or transmission of the pathogen. Thisincreases the risk of contraction for neonates and those unable to bevaccinated. Human efficacy data also indicates that the protection wanesby as much as 35% each year after immunization. Furthermore, strains ofB. pertussis are being clinically isolated do not express pertactin,which was originally characterized as one of the main virulence factorsof B. pertussis. For these reasons, there is a need for a new generationof effective pertussis vaccines as returning to a whole cell vaccine isnot an option due to the known risks.

Summary of Exemplary Embodiments

Various embodiments recite a vaccine composition including a B.pertussis antigen and an effective adjuvant amount of a high molecularweight glucose polymer, wherein the composition is administeredintranasally.

Various embodiments recite a vaccine composition including the B.pertussis antigens and an effective adjuvant amount of a β-glucan,wherein the β-glucan is selected from a group that includes curdlan,dextran, and baker's yeast beta-1,3/1,6-d-glucan.

Various embodiments recite a vaccine composition wherein the compositionfurther includes an adenylate cyclase toxin antigen, such as RTX.

Various embodiments recite a vaccine composition wherein the compositioninduces a Th1/Th17 immune response.

Various embodiments further recite a method of immunizing a host againstpertussis by administering intranasally to the host a vaccinecomposition including a B. pertussis antigen and an effective adjuvantamount of a high molecular weight glucose polymer.

Various embodiments further recite a method of enhancing the immuneresponse of an intranasally administered Bordetella pertussis antigenthat involves co-administering the antigen and a high molecular weightglucose polymer.

The present disclosure also describes a vaccine composition, comprisinga Bordetella pertussis antigen, and an effective adjuvant amount of ahigh molecular weight glucose polymer, where the high molecular weightglucose polymer has a molecular weight of between 68 kDal and 680 kDal.The high molecular weight glucose polymer may be soluble or dispersiblein water or aqueous base, and gellable in the presence of aqueous acid.The high molecular weight glucose polymer may be gellable in therespiratory system in the presence of acid and CO₂. The high molecularweight glucose polymer may be a beta-glucan, a 1,3-beta-glucan polymer,a 1,3-beta-glucan/1,4-beta-glucan copolymer, a1,3-beta-glucan/1,6-beta-glucan copolymer, or a mixture thereof.

In various embodiments, the vaccine composition contains a Bordetellapertussis antigen which may be an extracellular toxin, an adhesionprotein, an outer membrane protein, a receptor protein, fragmentsthereof, or mixtures thereof. The Bordetella pertussis antigen may be anextracellular pertussis toxin (PT), the adhesion proteins filamentoushemagglutinin (FHA) and fimbriae (FIM)), the outer membrane proteinpertactin (PRN), the siderophore receptor protein FauA, thexenosiderophore receptor protein BfeA, the hemophore receptor proteinBfuR, fragments thereof, or mixtures thereof. The Bordetella pertussisantigen may be the extracellular pertussis toxin (PT), the adhesionprotein filamentous hemagglutinin (FHA), the siderophore receptorprotein FauA, fragments thereof, or mixtures thereof.

In various embodiments, the composition is formulated for intranasaladministration; for parenteral administration by subcutaneous (SC)injection, transdermal administration, intramuscular (IM) injection, orintradermal (ID) injection; or for non-parenteral administration by oraladministration, intravaginal administration, pulmonary administration,ophthalmic administration, or rectal administration.

The current disclosure describes a vaccine composition for intranasaladministration to a patient, including a Bordetella pertussis antigen,and an effective adjuvant amount of a high molecular weight glucosepolymer, where the high molecular weight glucose polymer is configuredto adhere to the airway of a patient, by forming a gel in the presencein the presence of CO₂ and aqueous acid.

The current disclosure describes a method of immunizing a host againstpertussis by administering a vaccine composition including a B.pertussis antigen and an effective adjuvant amount of a high molecularweight glucose polymer intranasally to the host.

The current disclosure also describes a method of enhancing the immuneresponse of an intranasally administered B. pertussis antigen thatinvolves co-administering the antigen and a high molecular weightglucose polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

In order to better understand various embodiments, reference is made tothe accompanying drawings, wherein:

FIG. 1 shows iron and heme acquisition in B. pertussis. The model basedon B. pertussis alcaligin, enterobactin and heme acquisition systems,and their homologues in other bacterial species. OM/IM: outer/innermembrane. In parentheses: fold changes in expression in vivo vs. invitro.

FIG. 2A and FIG. 2B show structures of the xenosiderophore receptor BfeAand the hemophore receptor BfuR, respectively. Sequences correspondingto antigenic peptides of Table 1 are highlighted in color.

FIG. 3 illustrates a model of curdlan-DTaP immunization leading to Tcell polarization and production of antibodies that recognize andneutralize infecting B. pertussis.

FIGS. 4A, 4B and 4C illustrate the synergistic improvement of DtaPadministered by intraperitoneal injection (IP-DTaP) by inclusion oftoxoid adenylate cyclase antigen (RTX) and show viable B. pertussis inlungs of mice at 3 days post challenge, enhanced production ofanti-pertussis toxin due to inclusion of RTX, and decreased IL-6 due toinclusion of RTX, respectively.

FIGS. 5A, 5B, 5C and 5D illustrate the bacterial burden, lung IL-6production, lung neutrophil recruitment, and PT/FHA IL-17 production ofsplenocytes, respectively, in aP, wP, and IN-caP immunized mice.

FIGS. 6A to 6F illustrate localization of acellular pertussis vaccine inthe upper and lower respiratory system after IN vaccination. FIG. 6Ashows a schematic of the vaccine tracking protocol. CD-1 mice wereintranasally vaccinated with either fluorescent DTaP alone (IN-aP) orfluorescent DTaP with curdlan (IN-caP). Vaccine particle deposition inthe lungs and nasal cavity was measured at 0, 6, 12, 24, and 48 h afterimmunization. FIG. 6B shows a representative image of Alexa Fluorlabeled DTaP vaccine particles. FIG. 6C shows representative images ofnasal cavity fluorescence at 6, 12, and 24 h after vaccination withIN-aP or IN-caP. The region of interest used for fluorescencequantification is shown in blue. FIG. 6D shows fluorescence measurementsnormalized to PBS control at 6, 12, and 24 h (n=4). Results shown asmean±SEM of total radiant efficiency, *P<0.05. P values were determinedby multiple T-tests with Holm-Sidak post hoc test between IN-aP andIN-caP vaccinated mice. FIG. 6E shows representative plots at 12 hshowing live, single cells that are CD11b⁺DTaP⁺. FIG. 6C shows flowcytometric analysis of CD11b⁺ cells from the lung that contain or arebound to DTaP particles at 6, 12, 24, and 48 h post immunization.Results shown as mean±SEM, *P<0.05, ***P<0.001, ****P<0.0001 (n=4). Pvalues were determined by one-way ANOVA with Dunnett's post hoc testcomparing IN-aP immunized mice to control mock vaccinated mice.

FIGS. 7A to 7D show acellular pertussis vaccine particle localization.FIG. 7A shows representative images of flash frozen lung sections 6 hafter immunization with IN-aP and IN-cap. Fluorescent particles weredetected using a 660 laser. Samples were counter-stained with NucBlue(blue) and ActinGreen (green). FIG. 7B shows quantifying fluorescentDTaP particles in lung tissue by determining the percentage area ofparticles per field of view (n=3-4, with averages of three images perlung). Results are shown as mean±SEM, *P<0.05. FIG. 7C showsrepresentative images of paraffin embedded nasal cavity sections 6 hafter immunization with IN-aP or IN-caP. FIG. 7D shows quantifyingfluorescent DTaP particles in nasal tissue by determining the percentagearea of particles per field of view. (n=3-4, with averages of threeimages per lung). P values were determined by one-way ANOVA with Tukey'spost hoc test.

FIGS. 8A to 8D show production of anti-PT and anti-FHA IgG in seruminduced by intranasal immunization. ELISAs were used to compareserological responses from mice immunized through IN or IP routes tomock vaccinated mice. Total IgG serum antibody titers from immunized andchallenged mice were quantified at day 3 post challenge, where FIG. 8Aand FIG. 8B show anti-PT and anti-FHA IgG production, respectively.Serum IgG1 and IgG2 antibody titers against B. pertussis (FIGS. 8C and8D, respectively) were compared to mock vaccinated mice at day 3.Results are shown as mean±SEM, **P<0.01, ***P<0.001, P<0.0001 (n=3-8). Pvalues were determined by one-way ANOVA with Dunnett's post hoc testcompared to mock vaccinated mice.

FIGS. 9A and 9B show that intranasal immunization induces production ofanti-B. pertussis IgA in the respiratory system. ELISAs were performedusing a lung homogenate supernatant (FIG. 9A) and a nasal lavage fluid(FIG. 9B) from vaccinated and challenged mice at day 3 postimmunization. IgA titers were determined against whole-cell B. pertussisvaccine. Results are shown as averages of two independent experiments,represented on a log¹⁰ scale for lung and linear scale of nasal lavagewith mean±SEM (n=4-8). **P<0.0001. P values were determined by one-wayANOVA with Dunnett's post hoc test compared to mock vaccinated mice.

FIGS. 10A to 10D show that intranasal immunization decreases pulmonarypro-inflammatory cytokines during challenge. Analysis of cytokines fromsupernatant of lung homogenate at day 3 pc. Cytokines IL-6 (FIG. 10A),IFN-γ (FIG. 10B), IL-5 (FIG. 10C), and IL-17A (FIG. 10D) were quantifiedby electrochemiluminescence assay. Results shown as mean±SEM (n=4-8),*P<0.05, **P<0.01, *** P<0.001, P<0.0001. P values were determined byone-way ANOVA with Dunnett's post hoc test compared to mock vaccinatedmice. Bars connecting groups indicate values determined by two-tailedun-paired t-test. Upper and lower limits of detection shown as dash ordotted lines, respectively, if data points reached these limits.

FIGS. 11A to 11C show that intranasal immunization reduced neutrophilaccumulation in the lung and circulating neutrophils, but did notgenerate lung TRM population after B. pertussis challenge. FIG. 11Ashows the percentage of live, CD11b+Gr-1hi neutrophils from a singlecell suspension of the peripheral blood. FIG. 11B shows the percentagesof CD11b+Gr-1hi neutrophils in single cell lung homogenates. FIG. 11Cshows the percentage of CD4+ T cells that are CD62L-CD44+CD69+ isolatedfrom the lung at day 3 pc. Results shown as means±SEM, *P<0.05 **P<0.01,***P<0.001, P<0.0001 (n=4-8). P values were determined by one-way ANOVAwith Dunnett's post hoc test compared to mock vaccinated mice.

FIGS. 12A to 12F show that intranasal immunization reduced therespiratory B. pertussis bacterial burden. Analysis of bacterial burdenwas determined at days 1 and 3 pc. Bacteria were quantified by countingof serially diluted CFUs following immunization and challenge. CFUcounts were determined from lung homogenate (FIGS. 12A and 12B), tracheahomogenate (FIGS. 12C and 12D), and nasal lavage fluid (FIGS. 12E and12F). Results are mean±SEM (n=4-8, with four averaged technicalreplicates) from two independent experiments. *P<0.05, **P<0.01,***P<0.001, **P<0.0001. P values were determined by one-way ANOVA withTukey's post hoc test compared to mock vaccinated mice, or betweenconnected columns. The dashed line represents the lower limits ofdetection due to plating.

FIGS. 13A and 13B show that Pertussis patients and mice immunized withFauA peptides have anti-FauA peptide antibodies. FIG. 13A shows ELISAdetection of IgG anti-FauA in:

-   -   convalescent patient sera (n=23).    -   control patient sera (n=12).    -   mice vaccinated with FauA peptides (n=4); and    -   control mouse sera (n=4).

FIG. 13B shows ELISA detection of IgG specific to various individualFauA peptides in convalescent or control patient sera. Each dot on thegraph represents a different patient. Ctrl: control; ND: not detected;LDL: lower detection limit; UDL: upper detection limit.

FIGS. 14A to 14C show bacterial load in vaccinated mice three days afterchallenge by infection with B. pertussis.

FIGS. 15A to 15C show serum antibody titers in vaccinated mice threedays after challenge by infection with B. pertussis. FIG. 15D shows IL-6levels in vaccinated mice three days after challenge by infection withB. pertussis.

FIG. 16 shows a protocol for testing long-term protection againstpertussis by intranasal vaccination.

FIG. 17 shows serum antibody titers in vaccinated mice from 1 to 5months following vaccination.

FIG. 18 shows serum antibody titers in vaccinated mice three days afterchallenge by infection with B. pertussis and 6 months after vaccination,where mice were vaccinated following the protocol of FIG. 16.

FIG. 19A and FIG. 19B show the impact of intranasal vaccination with aP,aP-alum, aP-curdlan, and aP-β-glucan (shown as aP+IRI-1501) on thenumber of B. pertussis antibody-secreting cells in bone marrow.

To facilitate understanding, identical reference numerals have been usedto designate elements having substantially the same or similar structureor substantially the same or similar function.

DETAILED DESCRIPTION OF THE INVENTION

The description and drawings presented herein illustrate variousprinciples. It will be appreciated that those skilled in the art will beable to devise various arrangements that, although not explicitlydescribed or shown herein, embody these principles and are includedwithin the scope of this disclosure. As used herein, the term, “or”refers to a non-exclusive or (i.e., and/or), unless otherwise indicated(e.g., “or else” or “or in the alternative”). Additionally, the variousembodiments described herein are not necessarily mutually exclusive andmay be combined to produce additional embodiments that incorporate theprinciples described herein.

Vaccine-induced protection in acellular pertussis vaccine (aP) immunizedindividuals has been associated with a robust antigen-specific IgGresponse to the components of the aP vaccines. Likewise, whole cellpertussis vaccine (wP) immunization also results in antigen-specific IgGresponses; with the addition of a shift to a more diverse T cellresponse, inducing cell-mediated immunity. In the murine model,immunization through intramuscular (IM) and intraperitoneal (IP)administration has been well characterized demonstrating a Th1/Th17response from wP immunized mice, and a Th2 with weak Th17 mediatedresponse in aP immunized mice following B. pertussis challenge. However,these immunizations fail to induce the mucosal immune responses elicitedfrom natural infection. Protection correlates with tissue residentmemory T (T_(RM)) cells in the lung and nasal cavity of convalescentmice, that produce interleukin-17 (IL-17) and interferon-gamma (IFN-γ),although T_(RM) activity in pertussis is yet to be studied in humans.T_(RM) cells have been shown to persist in the respiratory tissue andexpand upon re-challenge of a convalescent mouse with B. pertussis, aswell as decrease bacterial burden upon adoptive transfer to naïve mice.More recently the expansion of this population has been observedfollowing immunization by wP, a live-attenuated wP vaccine, an outermembrane vesicle vaccine, and intranasal administration of an aP vaccinewith TLR9 and stimulator of interferon genes (STING) agonists.

The induction of a mucosal immune response to B. pertussis is associatedwith the production of secretory IgA antibodies (sIgA) in the nasalcavity. In humans previously infected with B. pertussis, IgA antibodieshave been isolated from nasal secretions. B. pertussis-specific IgAantibodies isolated from convalescent patients have been shown toinhibit adherence of B. pertussis to respiratory epithelial cells invitro, suggesting a protective role of IgA antibodies in mucosalimmunity.

Conventional aP or DTaP vaccine does not contain a strongpro-inflammatory adjuvant. This disclosure describes IN aP or DTaPimmunization alone, or with an additional pro-inflammatory adjuvant. Asuitable pro-inflammatory adjuvant is a high molecular weight glucosepolymer, which may be a beta-glucan, e.g., curdlan. Vaccines containingthe adjuvant curdlan, a 1,3-beta-glucan derived from Alcaligenesfaecalis, were formulated. This polysaccharide has immunostimulatoryproperties and forms a “sticky” gel at a neutral pH, or at an acidic pHin the presence of CO₂. The adjuvant has a relatively large particlesize with dimensions sufficient to produce an inflammatory response. Incertain embodiments, the high molecular weight glucose polymer may be amedium molecular weight glucan polymer with a molecular weight above 68kDal, or between 68 kDal and 2MDal, or between 68 kDal and 680 kDal;this high molecular weight provides sufficient size for the inflammatoryresponse from the soluble polymer. In various embodiments, the highmolecular weight glucose polymer may be a whole glucan particle purifiedfrom a yeast. Such whole glucan particles typically have a particle sizeof 3 to 10 m, 3.5 to 8 μm, or 4 to 6 μm, and may include residualproteins and/or lipids from yeast cells. The whole glucan particles mayinclude 70% to 85%, 72% to 83%, or 75% to 80% 1,3-linked glucose units;3% to 15%, or 5% to 10%, 1,6-linked glucose units; and 3% to 15%, or 5%to 10%, 1,3/1,6-linked glucose units. Whole glucan particles havesufficient size to trigger an inflammatory response from the polymer. Invarious embodiments, the glucose polymer may be conjugated to a protein,such as bovine serum albumin.

In another embodiment, the adjuvant includes particles having a minimumdimension of 3 μm, such as aluminum oxide particles. In anotherembodiment the majority of the adjuvant particles were between 3 to 10μm, 3.5 to 8 μm, or 4 to 6 μm.

Curdlan and other β-glucan polymers has been shown to bind to dendriticcells through the ligand Dectin-1, thereby inducing expression of NF-xBleading to a Th1/Th17 mediated immune response as well as production ofantigen-specific respiratory IgA antibodies and serum IgG antibodies.Dectin-1 is a receptor for β-glucans, which binds β-glucans and mediatesthe production of secretion of proinflammatory cytokines. Use of aβ-glucan as an adjuvant may stimulate an immune response in a patient.

As discussed in the present disclosure, the gel properties of curdlanfacilitate aP or DTaP localization in the upper respiratory tract. Asignificant reduction in bacteria burden is found followingadministration of intranasally administered aP or DTaP vaccines. Highserum and respiratory antibody responses were measured, followingintranasal administration of aP or DtaP, with and without curdlan.Mucosal vaccination with acellular vaccine containing a beta-glucan maybe a strategy for decreasing incidence of pertussis.

It has now been found that immunization with B. pertussis antigenstriggers a mucosal response similar to natural B. pertussis infection.Immunization may induce production of pertussis specific immunoglobulinsthat may: 1) mediate complement-dependent bacterial killing, 2) preventcolonization by blocking bacterial attachment, and 3) neutralize toxinsat the site of infection. In some embodiments, a vaccine composition ofthe invention may provide a longer lasting and more effective form ofthe whooping cough vaccine, leading to decreased incidence ofasymptomatic carriers and contraction by immune compromised and neonatalindividuals.

In various embodiments of the invention, the vaccine composition mayinclude B. pertussis antigens selected from a group that includesextracellular pertussis toxin (PT), the adhesion proteins filamentoushemagglutinin (FHA) and fimbriae (FIM), pertactin (PRN), andcombinations thereof. The vaccine composition may include fragments ofB. pertussis antigens selected from a group that includes PT, FHA, FIM,pertactin, and combinations thereof.

Additional proteins targeted for peptide vaccine development include thesiderophore receptor FauA, the xenosiderophore receptor BfeA, and thehemophore receptor BfuR, and fragments thereof.

Antigen proteins, including those described above, were selected for usein the vaccine composition based on the following criteria:

-   -   They are present on the surface of the organism, allowing for        surface recognition and opsonophagocytosis.    -   They have conserved sequences, with the sequence of the        corresponding gene having:        -   at least 95% similarity across the clinical isolates tested,            and        -   85% similarity, 90% similarity, 95% similarity, or 98%            similarity to corresponding genes in other Bordetella            species.    -   They are highly up-regulated during infection by B. pertussis.    -   They are important for virulence, in that mutation of the        antigen proteins negatively affects bacterial growth and        pathogenesis.

For example, FIG. 1 shows iron and heme acquisition in B. pertussis. Themodel of FIG. 1 is based on B. pertussis alcaligin, enterobactin andheme acquisition systems. As seen in FIG. 1, FauA and BfeA are receptorsfor iron-carrying proteins, and BfuR is a receptor for a heme-carryingprotein. These proteins are exposed on the outer membrane of B.pertussis, and are thus good candidates as antigens for a B. pertussisvaccine. Other proteins involved in iron or heme transport, like thetransport protein TonB and the heme-transporting proteins BhuT, BhuU,and BhuV are less suitable antigen candidates, as they are not exposedon the outer membrane.

Acellular pertussis vaccines include PRN, PT, and FHA antigens (aP). AsPRN and FHA antigens are harvested from the whole bacteria, theseantigens may contain the lipooligosaccharide (LOS) endotoxin from thebacteria. The LOS endotoxin, if present, may serve as an antigen, andinduce formation of antibodies against the B. pertussis bacteria,enhancing the formation of antibodies by the aP vaccine. In variousembodiments, the LOS antigen may be added to the aP vaccine as a fourthantigen to enhance formation of pertussis antibodies.

The pertussis vaccine may be formulated with an adjuvant foradministration. The adjuvant may be aluminum hydroxide (aP-alum), or abeta-glucan. In some cases, the beta-glucan may be a 1,3-beta-glucan(aP-beta-glucan) or curdlan (caP or aP-curdlan).

As noted above fragments of B. pertussis proteins may be used asantigens. For example, extracellular regions of the proteins may beprepared and used as antigens. A bioinformatics pipeline may be used toidentify the most immunogenic regions of proteins to be used as antigensfor vaccination. A 3D protein structure analysis is performed. Thestructure analysis may use known crystal structures for a proposedantigen protein. Alternatively, the structure analysis may use acomputational study to predict the protein structure. Structures of thexenosiderophore receptor BfeA and the hemophore receptor BfuR are shownin FIG. 2A and FIG. 2B, respectively.

The extracellular regions of the proposed antigen proteins areidentified, and their immunogenicity is predicted based on theirhydrophobicity and B cell epitope predictions. Using this approach, thesequences of various potential immunogenic regions were identified.Antigenic fragments based on these sequences were prepared. The antigenfragment peptides may then be modified by adding a cysteine residue onthe N-terminus, and by conjugating them to a carrier protein. Thecarrier protein may be, but is not limited to, Keyhole Limpet Hemocyanin(KLH), diphtheria toxoid Cross-Reactive Material 197 (CRM197), orrecombinant tetanus toxoid (rTTHc). Based on this approach, multiplepotential antigenic peptide fragments based on FauA, BfeA, and BhuR wereidentified. These peptide fragments are presented in Table 1.

TABLE 1 Antigen Fragments based on FauA, BfeA, and BhuR. SEQ ID NO.Peptide name Peptide sequence  1 FauA peptide 1 (275-309)CHSNGFGSGFPLFYSDGSRTDFNRSVANNAPW ARQD  2 FauA peptide 2 (409-441)CYAMVGPAPAIGSFFDWRRAHIQEPSWADTLSP A  3 FauA peptide 3 (516-535)CFQPQNARDTSGGILPPIK  4 FauA peptide 4 (567-584) CQVIPGSSIPGFPNMQASR  5FauA peptide 5 (617-633) CHFTTKDASGNPINTNHPRSLF  6FauA peptide 6 (658-680) CWQSRMYQAAASPRGNVEVEQDSYAL  7BfeA peptide 1 (226-257) CYNKTNPDARDINAGHANTSDNGNPSTAGRE GV  8BfeA peptide 2 (287-313) CNLFAGDTMNNANSDFSDSLYGKFTNAM  9BfeA peptide 3 (403-427) CAGTRQTYTGGAIGGTAPADRDPKSR 10BfeA peptide 4 (342-368) CNARQREGLAGGPEGAPTAGGYDTARLK 11BfeA peptide 5 (555-584) CDYRNKIVAGTDVQYRLANGARVLQWTNSGK 12BfeA peptide 6 (487-533) CYKAPNLYQSNPNYLLYSRGNGCLASQTNTNGCYLVGNEDLSPETSVN 13 BfeA peptide 7 (650-677)CTYYGKQEGPSTNVRTGVELNGDGRQTIS 14 BfeA peptide 8 (701-729)CSNLFDKQLYREGNASSAGAATYNEPGRAY 15 BhuR peptide 1 (336-374)CEYFKRRADLDQMYQQGAGTSYQYGANRTHE ETTRKRVSL 16 BhuR peptide 2 (283-316)CAGTRNGHDLDNRADTGGYGSKRSQPSPEDY AQNN 17 BhuR peptide 3 (398-438)CRLRLDSSQDARRTRDGRAYARPGDPYFYGYPS GPYGRSNSI 18 BhuR peptide 4 (466-513)CEWYGNRTEQYSDGYDNCPAIPPGTPAPMGPR LCDMLHTNQADMPRVKG 19BhuR peptide 5 (537-571) CLRYDHYEQKPQQGGGYQNNPNAGALPPSSS GGRFS 20BhuR peptide 6 (591-639) CGFGYRAPSATELYTNYGGPGTYLRVGNPSLKPETSKGWELGARLGDDQL 21 BhuR peptide 7 (654-685)CIDKNVPLGKGSPQWQPAWDGQYPLGVTGLA NR 22 BhuR peptide 8 (754-799)CTRRDDVQYPEASASARYADFQAPGYG

In one embodiment of the invention, the vaccine composition includes aB. pertussis antigen and an effective adjuvant amount of a highmolecular weight polymer of glucose, such as β-glucan, dextran and thelike. Preferred β-glucans include curdlan and baker's yeastbeta-1,3/1,6-d-glucan. Curdlan is a bacterial and fungal β-1,3-glucanthat binds to Dectin-1 receptors which are expressed on macrophages anddendritic cells.

The term “effective adjuvant amount” will be well understood by thoseskilled in the art, and includes an amount of a high molecular weightglucose polymer which is capable of stimulating the immune response tonasally administered antigens, i.e. an amount that increases the immuneresponse of a nasally administered antigen composition.

In another embodiment, the vaccine composition of the invention mayfurther be supplemented with an adenylate cyclase toxoid (ACT) which mayimprove efficacy of the vaccine composition by 1) generating anti-toxinantibodies against ACT, and 2) slowing vaccine-driven strain evolution.Suitable adenylate cyclase toxin antigens include purified repeats inthe toxin domain (RTX antigen).

The vaccine composition of the invention may also contain additionaladjuvants such as aluminum hydroxide.

The vaccine composition of the invention may be used as part of aprime-boost vaccine regimen. Conventional acellular pertussis vaccine(DTaP) is administered to human patients in five prime vaccinations atthe following ages: 2 months, 4 months, 6 months, 15 to 18 months, 4 to6 years. Periodic acellular pertussis vaccine boosts (TDaP) may beadministered at age 11, and subsequently as needed. In a murine model,mice may be vaccinated with an aP vaccine as a prime, and then be givena boost vaccine 21 days later.

In one embodiment, the vaccine composition of the invention may beformulated for intranasal administration.

In other embodiments, the vaccine composition of the invention may beadministered using alternative routes of administration including,without limitation, parenteral administration methods, such assubcutaneous (SC) injection, transdermal, intramuscular (IM),intradermal (ID), as well as non-parenteral, e.g., oral, intravaginal,pulmonary, ophthalmic and or rectal administration.

Pertussis toxin (PT) is an essential virulence factor, responsible formultiple factors in the pathogenesis of B. pertussis. PT facilitatesinfection by aiding in adherence to ciliated airway epithelial cells andthrough disruption of host innate immune cell recruitment to the site ofinfection. In numerous studies it has been demonstrated thatneutralization of PT alone ablates symptoms of the disease.Neutralization of pertussis toxin at the site of infection may inhibitthe systemic long-range activity of PT before colonization of therespiratory tract. Intranasal immunization with pertussis antigens mayprime a protective systemic and mucosal immune response. Furthermore,the gel-like properties of curdlan may have a beneficial role inincreasing antigen uptake.

EXAMPLES

The vaccines administered were prepared no longer than 1 h beforeadministration. In Examples 1-3, INFANRIX (GSK) human vaccine (DTaP),and the National Institute for Biological Standards and Control WHOwhole-cell pertussis vaccine (NIBSC code 94/532) were used as the aPacellular pertussis vaccine. The aP vaccine used in Examples 1-3contains DTaP with formaldehyde killed pertussis toxoid. In Examples4-8, genetically detoxified pertussis toxoid was used. The aP vaccine inExamples 4-8 contained PRN and PT antigens obtained from ListBiologicals and FHA antigen obtained from ENZO bio. As the PRN and FHAantigens are harvested from B. pertussis bacteria, they may also containthe lipooligosaccharide (LOS) endotoxin from the bacteria.

Vaccines administered with curdlan were diluted with PBS to 1/12^(th) ofthe human dose (based on total antigen content). Curdlan adjuvant wasadministered at 200 μg per mouse. A vaccine dose of 1/12^(th) the humandose was the highest concentration of vaccine that was possible to usedue to volume of vaccine required for the solubility of curdlan.

Curdlan (Invivogen, tlrl-curd) was prepared by dissolving 50 mg in 2.5ml sterile purified water. Curdlan preparation was brought into solutionby adding 100 μl 1N NaOH and vortexing. The curdlan suspension (20mg/ml) was then sonicated for 10 mins and placed in 37° C. water bathuntil administration. At the time of vaccination, the vaccinescontaining curdlan were administered in a liquid form to reduce risk ofchoking due to excessive gel formation in the airway. IN administeredmice recovered at the same rate as IP immunized mice. These experimentswere conducted in accordance with the National Institutes of HealthGuide for the care and use of laboratory animals. The protocols usedwere approved by West Virginia University Institutional Animal Care andUse Committees (WVU-ACUC protocol 1602000797). Curdlan, as used hereinis a medium molecular weight glucan polymer, i.e., a polymer with amolecular weight of between 68 kDal and 680 kDal. The 1,3-β-glucanadjuvant, as used herein, is a whole glucan particle adjuvant having aparticle size of 3 to 4 μm.

As comparison models, a group of mice were mock vaccinated withphosphate buffered saline (PBS). The mock vaccinated group was dividedinto a control group (Control or NVNC) which was not infected with B.pertussis, and a group which was infected with B. pertussis (Mock Vac).A third group of mice included unvaccinated mice recovering from B.pertussis infection (Convalescent)

Example 1 Development of a Model to Evaluate ACT as an Antigen forInclusion in aP

aP vaccine lots are validated based on using high doses such as ⅕^(th)human dose with intranasal B. pertussis challenge. However, those dosesare not physiologically relevant to a mouse. Consequently, high doses ofDTaP or Tdap antigens do not afford the ability to determine if additionof new antigens to the “base” vaccine can improve protection. In orderto circumvent these issues, an approach was designed that could identifyantigens that synergistically improve a vaccine. In these studies, 30variables of data per mouse are collected to determine the correlates ofprotection. Lung IL-6 levels are an indication of 1) inflammation due topresence of pathogen and 2) ACT activity is known to activate IL-6secretion. The vaccine minimum protective dose was identified as1/40^(th) based on the viable bacteria (FIG. 4A). 1/80^(th) was selectedas the dose to test inclusion of RTX antigen because of the amounts ofviable bacteria in the respiratory tract (FIG. 4A), high IL-6 (FIG. 4B),and low total anti-PT (FIG. 4C).

Inclusion of RTX Antigen into the aP

Mice immunized with RTX alone with aluminum hydroxide are not protectedagainst Bp challenge. However, when the 1/80^(th) aP vaccine wassupplemented with RTX antigen (5 μg), a significant decrease inbacterial burden was observed at day 3 (FIG. 4A) compared to 1/80^(th)IP-aP alone. RTX inclusion also decreased IL-6 (FIG. 4B) which suggeststhat immunization neutralizes AC toxin activity in the murine host. Highamounts of serum IgG were also detected that recognizes RTX using ELISA,confirming the immunization induced RTX-specific antibody production.Surprisingly, it was observed that inclusion of RTX enhanced anti-PTproduction as well (FIG. 4C) demonstrating potential adjuvant effects ofRTX.

Example 2 Model of IN-caP Intranasal Immunization

For the studies, curdlan was selected as an adjuvant in order to inducea Th1/Th17 response which would mimic the wP or convalescent immunity.By interacting with the Dectin-1 receptor, curdlan primes naïve CD4+ Tcells to differentiate to Th1 and Th17 T cells. In addition to itsexcellent adjuvant properties, curdlan can form a gel in the presence ofcarbon dioxide and at acidic pH. Thus, intranasal immunization withcurdlan allows the antigen to adhere to the airway to allow uptake ofthe antigens by antigen presenting cells.

In the model presented in FIG. 3, curdlan encases the aP particles andfacilitates their presentation to the mucosa as well as uptake of theantigens by antigen-presenting cells such as dendritic cells (DC).

In order to observe if curdlan facilitates the localization of thevaccine to the upper airway, DTaP was fluorescently labeled with Alexafluor 660 dye (FIG. 3). It was observed that when curdlan was added asthe adjuvant, three-fold more fluorescent signal was detected in thenasal flushes of mice compared to immunization without curdlan. The datasuggests that curdlan gel facilitates adherence of the vaccine in theupper airway.

IN-caP Intranasal Immunization Protects Against B. pertussis Challengein Mice

Mice were immunized with curdlan alone (200 μg/dose) with no antigensadded. When mice were challenged with Bp, no protection was observed(FIG. 3A; curdlan only group). A 1/12^(th) human dose of DTaP (aPvaccine) was formulated and supplemented with curdlan (200 μg/dose).Intranasally administered curdlan-adjuvanted aP (IN-caP) immunized micewere then challenged with Bp. A decreased bacterial burden in the lungwas observed 1 and 3 days post infection by over 3 logs (FIG. 3A).Limited protection was observed in the intranasally administered aP(IN-aP, aluminum hydroxide adjuvant), indicating that curdlan adjuvantfacilitates induction of an adaptive response with IN immunization ofaP. Compared to naïve mice, IN-caP immunized mice had reduced IL-6 (FIG.3B) as well as low amounts of total neutrophils in the lung (FIG. 3C),which suggests that IN-caP immunization is protecting the mice from B.pertussis challenge.

IN-caP Immunization Results in FHA/PT Antigen-Specific IL-17 ProducingSplenocytes

The IN-caP vaccine contained curdlan and aluminum hydroxide adjuvants.To determine the T-cell response, Elispot analysis was performed onsplenocytes of the immunized mice. Splenocytes were stimulated with PTand FHA antigens and it was observed that mice immunized with IN-caPinduced significantly more IL-17-producing splenocytes than wP immunizedmice (FIG. 3D). The data suggests that IN-caP induces a Th1/Th17response.

TABLE 2 Analysis of antibody production in IN immunized mice. Bp FHA PTLOS Vaccine challenged IgG IgG IgG none NO − − − PBS YES − − − IP-aP(1/5th human dose) YES +++ +++ − IN-caP (1/12th human YES +++ +++ −dose) IP-wP (1/5th human dose) YES +++ − +++ YES IN-cwP (1/12th humanYES + − ++ dose)

To determine the antibody response triggered by IN-caP immunization, IgGproduction against FHA, PT, and lipooligosaccharide (LOS) antigens weremeasured in each vaccine group by ELISA. As seen in Table 2, both IP-aPand IN-caP induced production of FHA and PT IgGs, but did not induceproduction of LOS IgG. Intraperitoneally administered whole cellpertussis vaccine (IP-wP) and an intranasal whole cell pertussis vaccinecontaining curdlan (IN-cwP) induced production of FHA and LOS IgGs, butdid not induce production of detectable anti-PT. Additionally, theimmune response against FHA induced by IN-caP was stronger than theimmune response against FHA induced by IN-cwP.

Example 3 Vaccination of Mice for Vaccine Particle Tracking

CD-1 (outbred; strain code 022) mice aged four weeks were obtained fromCharles River Laboratories. At five weeks mice were anesthetized with 77mg/kg ketamine and 7.7 mg/kg xylazine. Mice were administered 50 μl ofvaccine or control, with 25 μl into each nostril (IN).

Tracking of DTaP in Respiratory System

DTaP vaccine particles were labeled using the Alexa Fluor 660 ProteinLabeling Kit (Molecular Probes). Briefly, 0.5 ml of DTaP vaccine wasadded to 50 μl of 1 M sodium bicarbonate, then added to Alexa Fluor 660dye stock. The mixture was incubated for 1 h at room temperature withagitation. The solution was concentrated by dialysis inphosphate-buffered saline overnight to remove unlabeled dye. Vaccineparticles were examined using the Cy5 channel on an EVOS FL microscope.Particles were mounted on a slide and visualized using a 100× objective.Particle diameter was measured using ImageJ (version 1.52a) with theline segment tool in proportion to the scale bar. Four fields of viewwere measured to determine particle size and standard deviation. Labeledvaccine was used to immunize mice. At 0, 6, 12 and 24 h postvaccination, fluorescent signal was measured using an IVIS Spectrum(Xenogen), as shown in FIG. 6A. Mice were anesthetized using 3%isoflurane, mixed with oxygen prior to and throughout imaging. Thefollowing parameters were used: 1) A binning setting of 4 was keptconstant for all images, 2) each image was quantified using theautomatic image setting, 3) fluorescence photons were measured usingtotal radiant efficiency of a common region of interest placed on thenasal cavity, and 4) measurements were normalized using Living Image(Xenogen ver. 2.5). Use of this quantification method accounted forvariations in exposure between images.

At 6, 12, 24, and 48 h post vaccination, animals were euthanized toquantify DTaP in lungs by flow cytometry analysis, as shown in FIG. 6A.Lungs were removed, and homogenized using gentleMACS C tubes (MiltenyiBiotec) with enzymatic lung dissociation kit (Miltenyi Biotec,130-095-927). All samples were blocked using Fc Block (BD), thenlabelled with Alexa Fluor 700—conjugated CD11b (Biolegend, 101222), DTaPparticles were detected with Cy5 channel. Following a 1 h darkincubation labeled samples were washed, then fixed using 0.4% w/vparaformaldehyde overnight. Samples were resuspended in PBS and analyzedon an LSR Fortessa flow cytometer (BD). DTaP containing myeloid cellswere classified as CD11b⁺DTaP⁺ single, live cells.

Detection of DTaP Particles in Lung and Nasal Cavity

Detection of DTaP particles in the lung and nasal cavity were confirmedusing confocal imaging. Mock vaccinated and challenged mice wereeuthanized at 6 h post challenge, as shown in FIG. 6A. Prior tohomogenization, the post-caval lobe of the mouse lung was removed. Thepost-caval lobe was flash frozen in OCT medium (Tissue Plus, FisherHealthcare), using liquid nitrogen. The samples were stored at −80° C.until sectioning. Sectioned samples (6 μm) were fixed in acetone, thenstained with ActinGreen Ready Probes (Invitrogen) and NucBlue ReadyProbes (Invitrogen), using manufacturer protocols.

Skulls were removed from mouse, and the lower jawbone discarded. Theskulls were fixed in formalin for 12 h at 4° C., then de-calcified atroom temperature for 24 h, before samples were embedded in paraffin.Sectioned samples were de-paraffinized and rehydrated using xylene, andwashes with decreasing ethanol concentrations (100 to 70%). An antigenretrieval step was performed using citrate buffer, where samples wereheated to 98° C. for 20 mins. Samples were then stained as mentionedabove. Samples were analyzed for DTaP particles in tissue and airwaymucus using a Nikon AiR confocal microscope. Images were acquired usingDAPI, FITC, and Cy5 channels using a 100× oil immersion lens (100×/1.40Nikon Plan Apo). DTaP particles were quantified using ImageJ. Briefly,the threshold tool was used to select only the fluorescent particlesabove background levels. Then, the threshold adjusted area wasquantified using the analyze particles tool. Thus, the data isrepresented as the percentage of fluorescent particles per area of thetotal image field. Three image fields per sample were quantified andaveraged per mouse.

B. pertussis Strains and Growth Conditions

B. pertussis strain UT25Sm1 was used for murine challenge in allexperiments. UT25Sm1 was cultured on Bordet Gengou agar plus 15%defibrinated sheep's blood (Remel) with streptomycin 100 mg/ml. B.pertussis was incubated at 36° C. for 48 h, then transferred to modifiedStainer-Scholte liquid medium, without the cyclodextrin, heptakis.Liquid cultures were incubated for 24 h at 36° C., with shaking untilreaching an OD₆₀₀ of ˜0.6, at which time cultures were diluted forchallenge dose.

Vaccination and B. pertussis Challenge

IN immunized mice received 50 μl of vaccine as described above. IPimmunized mice received 200 μl of vaccine injected into the peritonealcavity. IN and IP immunized mice received the same antigen dose of1/12^(th). Mice received a boost of the vaccines with the sameconcentrations twenty-one days after initial immunization. Atthirty-five days post initial vaccination, mice were challenged with2×10⁷ CFU B. pertussis administered in 20 μl through nostrils. At days 1and 3 post challenge (pc), mice were euthanized, blood and respiratorytissue were isolated as previously described.

Serological Analysis of B. pertussis Specific Antibodies

Serological responses specific to B. pertussis antigens were quantifiedby ELISA. High-binding microtiter plates were coated with PT (50ng/well)(LIST Biologicals) and FHA (50 ng/well) (Enzo Life Sciences), asdescribed in Boehm et al. Serological responses against UT25Sm1 werecultured to an OD₆₀₀ of 0.24 and microtiter plates coated with 50 μl ofbacteria per well. Bound antibodies were detected using goat anti-mouseIgG, IgA, IgG2a, and IgG1 antibody conjugated to alkaline phosphatase(Southern Biotech). Positive antibody titers were determined using abaseline set at two times the average of blanks.

Quantification of Pulmonary and Blood APCs

To determine cell types infiltrating the lung and leukocytes present inperipheral blood, single cell suspensions from the tissues mentionedabove were prepared. Briefly, lung tissue was homogenized by Douncehomogenizers, filtered with a 100 μm filter, and red blood cells werelysed for 2 min at 37° C. (Pharmlyse). Single cell populations wereblocked by initial incubation with Fc Block (BD, 553141) for 15 min at4° C. Cell populations were incubated in the dark with antibodies tocell surface markers for 1 h at 4° C. Neutrophil populations wereidentified using: PE-conjugated GR-1 (BD, 553128), Alexa Fluor700-conjugated CD11b (Biolegend, 101222). Neutrophils were classified asCD11b+Gr-1^(hi) single, live cells. TRM populations were determinedusing: APC-Cy7-conjugated CD4 (Biolegend, 100526), BB515-conjugated CD44(BD, 564587), APC-conjugated CD62L (BD, 553152), and BV421-conjugatedCD69 (BD, 562920).

Lung Homogenate Cytokine Analysis

To quantify inflammatory cytokines at the site of infection, lunghomogenate supernatant was prepared and stored at −80° C., as describedin prior work. Quantitative analysis of cytokines was performed usingMeso Scale Discovery cytokine kits: V-PLEX pro-inflammatory panel(K15048D) and IL-17A V-PLEX (K152RFD), per the manufacturer'sinstructions.

Statistical Analysis

Experiments in the study were performed with 3 to 8 biologicalreplicates. Data were analyzed using GraphPad Prism 7. ROUT method wasused to removed outliers. Comparisons between groups were performedusing one-way analysis of variance (ANOVA) with Dunnett's and Tukey'spost hoc tests. Comparisons between groups with or without curdlan wereanalyzed by two-tailed unpaired t-test, when applicable multiple T-testswith Holm-Sidak post hoc test were applied to curdlan inclusion groups.

Acellular Pertussis Vaccine was Retained in the Upper and LowerRespiratory Tract when Administered by Intranasal Administration.

To determine if use of curdlan would increase vaccine retention in therespiratory system, CD-1 mice were intranasally (IN) vaccinated withcommercially available DTaP (IN-aP), DTaP with curdlan (IN-caP), orphosphate-buffered saline (PBS; mock vaccinated) and the vaccine for upto 48 h after vaccination. The protocol is illustrated in FIG. 6A. Tovisualize vaccine presence in the respiratory system, DTaP vaccineparticles were labeled with a fluorescent fluorophore (FIG. 6B). Thesize of the labeled particles was measured, and determined to be1.52±0.76 μm, on average. Using in duo animal imaging, we observedfluorescently labeled DTaP particles in the nasal cavity at 6, 12 and 24h post-vaccination (FIG. 6C). At 12 h post-vaccination, significantlyhigher levels of fluorescence were detected in IN-caP vaccinated mice,compared to IN-aP mice (FIG. 6D). This suggests more DTaP particles wereretained in the nasal cavity. This method resulted in the quantificationof total particles in nasal cavity.

To quantify DTaP particles that were bound to innate immune cells flowcytometry was utilized. Single-cell suspensions were prepared fromhomogenized lung tissue and antigen presenting cells (APCs) bound toDTaP were quantified as live, single cells positive for CD11b⁺DTaP⁺(FIG. 6E). A significant increase in CD11b⁺ cells that were bound to orcontained DTaP particles was observed in IN-aP mice, compared to IN-caP(FIG. 6F). Together, these data suggest a higher deposition of DTaP inthe lung with IN-aP when compared to IN-caP. Conversely, in the nasalcavity higher levels of DTaP was measured when mice were vaccinated withIN-caP, compared to IN-aP. These findings suggest that addition ofcurdlan to the DTaP vaccine causes retention in the nasal cavity, butwithout it, the vaccine components are more readily detected in thelung.

To visualize the deposition of DTaP particles, sections from the lungand nasal cavity were imaged using confocal microscopy. Vaccinated micewere euthanized after 6 h. Lung tissue was flash frozen, and skulls wereembedded in paraffin for sectioning. Sections from the lung and nasalcavity were counterstained with NucBlue and ActinGreen to visualizeepithelial tissue and fluorescent DTaP particles (FIG. 7A and FIG. 7C).Vaccine particles were quantified by measuring the percentage of totalimage field emitting DTaP fluorescence. A significant increase offluorescent particles in the lungs of mice that were vaccinated withIN-aP was detected, compared to mice vaccinated with IN-caP (FIG. 7B).Using microscopy, there was no significant difference in the number ofparticles detected in the nostrils when comparing mice vaccinated withIN-aP to mice vaccinated with IN-caP (FIG. 7D). Interestingly, DTaPparticles from the IN-aP vaccinated mice were localized in the lumen ofthe nasal passages, while particles from IN-caP vaccinated mice weredeposited into the epithelial cells (FIG. 7C). Overall, these datasuggest curdlan impacts localization of DTaP in the airway.

Example 4

Vaccination of Mice for with Genetically Detoxified Pertussis Toxoid

This example, and all subsequent examples, were carried out withgenetically detoxified pertussis toxoid vaccines. PRN and PT antigenswere obtained from List Biologicals and FHA antigen was obtained fromENZO bio.

The following vaccines were formulated:

-   -   aP vaccine: PRN, PT, and FHA antigens.    -   aP-alum: PRN, PT, and FHA antigens, with an aluminum hydroxide        adjuvant.    -   aP-curdlan: PRN, PT, and FHA antigens, with a curdlan adjuvant,        where the curdlan adjuvant is a medium molecular weight glucose        polymer.    -   aP-β-glucan: PRN, PT, and FHA antigens, with a 1,3-β-glucan        adjuvant, where the 1,3-β-glucan adjuvant is a whole glucan        particle adjuvant having a particle size of 4 to 6 μm.

In aP-alum, the aluminum hydroxide adjuvant is an aluminum hydroxide wetgel suspension. The aluminum hydroxide induces a Th2 response byimproving the attraction and uptake of antigen by antigen-presentingcells (APCs). Aluminum hydroxide particles have a net positiveelectrical charge at pH 5-7, and are attracted to negatively chargedantigens.

CD-1 mice aged weeks were anesthetized with 77 mg/kg ketamine and 7.7mg/kg xylazine. Mice were administered 50 μl of vaccine, with 25 μl intoeach nostril (IN). Four groups of mice (n=4) were vaccinated, with eachgroup being vaccinated with one of the aP, aP-alum, aP-curdlan, andaP-β-glucan vaccines. Each vaccinated group of mice was given a boostvaccination 3 weeks later. At 10 weeks of age, the mice were challengedby exposure to B. pertussis. A fifth group of mice was mock-vaccinatedwith PBS, then challenged by exposure to B. pertussis. A sixth group ofmice (NVNC) was mock-vaccinated with PBS, without being challenged by B.pertussis. Finally, a group of unvaccinated mice recovering fromexposure to pertussis was examined (Convalescent mice).

To determine if an IN-delivered pertussis vaccine would induce asystemic immune response, enzyme-linked immunosorbent assays (ELISAs)with serum from vaccinated and challenged mice against B. pertussisantigens found in the vaccine were performed. Pertussis toxin (PT) andfilamentous hemagglutinin (FHA) antigens were tested. ELISAs were notperformed against pertactin antigen, as 85% of current clinical isolatesin the US do not express the protein. Serum anti-PT IgG titers weresimilar between mice immunized with IP-aP and those immunized through INadministration, as no significant differences were determined betweenIP-aP, IN-aP or IN-caP (FIG. 8A). A robust titer response to thebacterial adhesin FHA in IN vaccinated mice was observed. However, IN-aPserum anti-FHA titer was 7-fold higher than IN-caP (FIG. 8B). Inductionof IgG1 antibodies (FIG. 8C) and IgG2a antibodies (FIG. 8D) by INadministration of DTaP (with or without curdlan) was studied to see ifIN administration would lead to an increased Th1 immune response,resulting in a higher ratio of IgG2a compared to IgG1 antibodies.Neither the intranasal nor intraperitoneal route impacted the ratio ofIgG2a to IgG1. Neither use of an aluminum hydroxide adjuvant (aP) nor acurdlan adjuvant (caP) impacted the ratio of IgG2a to IgG1.

IgA antibodies and a local mucosal immune response in the lung and nasalcavity are important to B. pertussis immunization. The presence of IgAantibodies in the murine respiratory tissue due to IN immunization wasmeasured. Using ELISAs, B. pertussis-specific IgA titers in homogenizedlung tissue supernatant and nasal lavage fluid were measured. A robustIgA response was observed in the lung only when mice were immunizedthrough the IN route (FIG. 9A). Similar results were not observed afterintraperitoneal immunization. Antigen-specific IgA response was also notobserved in mice immunized with curdlan alone. Detectable IgA B.pertussis-titer was observed in the nasal lavage fluid from both IN-aPand IN-caP vaccinated groups, although only IN-aP resulted in asignificant increase compared to baseline levels (FIG. 9B). The presenceof B. pertussis binding IgA in the lungs and nasal cavity suggests thatIN DTaP is capable of priming a mucosal immune response in the upper andlower murine respiratory systems.

Neutralization of PT and inhibition of bacterial adhesins associatedwith DTaP protection leads to a reduced pro-inflammatory environment atthe site of infection when compared to a natural infection. Conversely,challenge in whole-cell protected animals resulted in a severepro-inflammatory response, similar to the natural infection of B.pertussis. To determine whether the immunostimulatory properties ofcurdlan would induce a more pro-inflammatory response followingchallenge with B. pertussis compared to IP-aP, cytokine concentrationswere determined from supernatant of lung homogenate at day 3 postchallenge. There is a significant reduction of IL-6 in the lungs ofeither IN-aP or IN-caP immunized mice when compared to mock vaccinatedor IN-curdlan control mice, as shown in FIG. 10A. IL-6 levels arecomparable to levels observed in IP immunized groups. A similarreduction in the Th1-associated cytokine IFN-γ and the cytokine IL-17Awas observed in the IN administered groups, IN-aP and IN-caP; however,these levels were higher in IN-caP immunized mice (FIGS. 10B and 10D). Asignificant increase in the Th2-associated cytokine interleukin-5 andIL-17A was observed in IP-wP immunized mice, compared to mock vaccinatedmice (FIGS. 10C and 10D). The whole cell vaccine (wP) also did notsignificantly reduce IL-6 or IFN-γ.

IN administration of curdlan, and moreover, vaccine administrationthrough the IN route regardless of adjuvant has been shown to induce anincreased IL-17 response. Levels of IL-17 in mice immunized with IN-caPwere observed and compared to those in mice immunized with IN-aP orIP-aP. IL-17A in the lung supernatant was quantified, and significantincreases of IL-17A with the addition of curdlan in IP-caP and IN-caPimmunized mice were observed. When compared to IP-aP and IN-aP groups,IL-17A levels increased 4-fold and 14.9-fold, respectively (FIG. 10D).Furthermore, IN administration induced a significant increase in IL-17Acompared to IP immunization. This IL-17A response was lower than therobust IL-17A induced by IP-wP (FIG. 10D).

Natural infection with B. pertussis causes severe leukocytosis, whichcan be measured by elevated neutrophils in the peripheral blood.Following B. pertussis challenge, all vaccinated groups showedameliorated symptoms of leukocytosis by day 3 pc; however, only theadministration of DTaP either by IP or IN administration significantlyreduced CD11b⁺Gr-1^(hi) neutrophils in the peripheral blood (FIG. 11A).In the lungs, neutrophils were decreased in IN-aP, IN-caP, and IP-aPimmunized groups compared to mock vaccinated mice (FIG. 11B).

To determine if IN-aP or IN-caP could induce the expansion of a T_(RM)population, population, CD4 T cells were isolated from the lung at day 3pc, and were identified as T_(RM) cells based on expression of surfacemarkers: CD4+CD62L-CD44+CD69+. We did not observe a statisticaldifference of this population following challenge with either IP or INadministered vaccines. However, we did observe a slight increase inIP-wP, IP-caP, IN-aP and IN-caP compared to mock vaccinated mice (FIG.6c ). Taken together these data suggest that immunization with DTaPthrough the IN route reduces the pro-inflammatory environment of themurine lung during B. pertussis challenge in a manner similar toIP-aP-mediated protection.

Lastly, the clearance of B. pertussis from the respiratory tractfollowing IN immunization was examined. At days 1 and 3 pc, viablebacterial burden was quantified by counting of CFU in the lung, trachea,and nasal lavage fluid. A significant reduction in viable bacteriarecovered from the lung was observed in all immunized groups by day 3pc; however, these changes were not observed at day 1 pc (FIGS. 7A and7B). In IN-aP and IN-caP immunized mice, bacterial burdens were reducedby 99.4% and 99.7%, respectively, compared to mock vaccinated mice. Thisreduction in viable bacterial burden was superior to that of miceimmunized by IP-aP, an immunization that is known to be effective (FIG.7B). This reduction in bacterial burden was not observed followingimmunization with the negative control (IN-curdlan), suggesting anantigen-specific response. Similar trends were observed in the tracheahomogenate (FIGS. 7C and 7D), and in nasal lavage fluid (FIGS. 7E and7F), as all immunized groups regardless of IP or IN delivery weresignificantly reduced compared to mock vaccinated mice. In summary, weobserved similar clearance of B. pertussis from the respiratory tract ofmice immunized intranasally, compared to mice immunized with vaccinesknown to be protective by the IP route.

Example 5

Pertussis patients and mice immunized with FauA peptides have anti-FauAantibodies. Mice were vaccinated with a set of six FauA-derived peptides(n=4), specifically SEQ ID NOS. 1 to 6 of Table 1; the peptides wereconjugated to CRM197, a non-toxic mutant of diphtheria toxin. A set ofunvaccinated control mice (n=4) was also tested. FIG. 13A shows ELISAdetection of IgG antibodies in mice vaccinated with FauA peptides (n=4),where the antibodies bind to peptides having one of SEQ ID NOS. 1 to 6.These antibodies are not found in unvaccinated control mice. FIG. 13Aalso shows that convalescent pertussis patients (n=23) have sera whichcontain FauA antibodies which recognize FauA-derived peptides having SEQID NOS. 1 to 6. Again, these antibodies are not found in sera fromcontrol patients (n=12).

FIG. 13B shows ELISA detection of IgG which bind the individual FauApeptides of SEQ ID NOS. 1 to 6 in convalescent or control patient sera.The control patients did not have antibodies which recognized the FauApeptides. Antibodies which recognized each FauA peptide of Table 1 (SEQID NOS. 1 to 6) were detected in the convalescent patients.

Example 6 Vaccination of Mice for Vaccine Particle Tracking

CD-1 (outbred; strain code 022) mice aged four weeks were anesthetizedwith 77 mg/kg ketamine and 7.7 mg/kg xylazine. Mice were administered 50μl of PBS control or an aP vaccine with PT, FHA, and PRN antigens, with25 μl into each nostril (IN). After 21 days, a similar boost vaccine wasadministered into each nostril. Mice were divided into the followinggroups (n=4 for each group):

-   -   NVNC: PBS Control;    -   Mock-Vac: PBS Control;    -   aP: aP vaccine;    -   aP-alum: aP vaccine with an aluminum hydroxide adjuvant;    -   aP-curdlan: aP vaccine with a curdlan adjuvant;    -   aP-β-glucan: aP vaccine with a 1,3-beta-glucan adjuvant; and    -   Convalescent: Unvaccinated mice recovering after a B. pertussis        infection.        With the exception of the convalescent mice and NVNC mice, each        group of mice was challenged by infection with B. pertussis 35        days after administration of the initial intranasal vaccination.

Three days post challenge, the respiratory track bacterial burden wasmeasured for each group of mice except the unchallenged NVNC mice.Burden was measured by nasal lavage, and in the lungs and trachea, usingtechniques described above. Results are shown in FIGS. 14A to 14C. Useof a curdlan or 1,3-beta-glucan adjuvant produced similar results to useof an alum adjuvant.

Three days post challenge, total IgG serum titers to B. pertussis andIgG serum titers to the PT and FHA vaccine antigens were measured, andfound to be significantly elevated after challenge in mice vaccinatedintranasally with aP, aP-alum, aP-curdlan, and aP-β-glucan vaccines.Results are shown in FIGS. 15A, 15B, and 15C. Mice in the NVNC andMock-Vac groups showed no antibodies to B. pertussis (FIG. 15A) or tothe PT or FHA vaccine antigens (FIGS. 15B and 15C). Convalescent miceshowed antibodies to B. pertussis, but to a lesser extent than any ofthe vaccinated groups (FIG. 15A). Convalescent mice showed no antibodiesto the PT vaccine antigen (FIG. 15B), although they did show FHAantibodies.

Three days post challenge, production of the cytokine IL-6 in micevaccinated intranasally with aP, aP-alum, aP-curdlan, and aP-β-glucanvaccines was comparable to IL-6 production in the unchallenged NVNCmice, as shown in FIG. 15C. Production of the cytokine IL-6 in Mock-Vacand convalescent mice was substantially higher than in any of thevaccinated groups of mice.

Example 7: Long-Term Pertussis Protection

A protocol for testing long-term protection against pertussis byintranasal vaccination is shown in FIG. 16. CD-1 (outbred; strain code022) mice aged four weeks were anesthetized with 77 mg/kg ketamine and7.7 mg/kg xylazine. Mice were administered 50 μl of PBS control or an aPvaccine with PT, FHA, and PRN antigens, with 25 μl into each nostril(IN). After 21 days, a similar boost vaccine was administered into eachnostril. Levels of B. pertussis antibodies were measured 30 days afterthe initial prime vaccination, and at 30-day intervals thereafter (FIG.17). Mice were divided into the following groups (n=4 for each group):

-   -   NVNC: PBS Control;    -   Mock-Vac: PBS Control;    -   aP: aP vaccine;    -   aP-alum: aP vaccine with an aluminum hydroxide adjuvant;    -   aP-curdlan: aP vaccine with a curdlan adjuvant;    -   aP-β-glucan: aP vaccine with a 1,3-beta-glucan adjuvant; and    -   Convalescent: Unvaccinated mice recovering after a B. pertussis        infection.

As shown in FIG. 17, the prime-boost vaccination protocol significantlyincreased levels of antibodies against B. pertussis in intranasallyvaccinated mice for at least five months post-vaccination, when comparedto NVNC mice or Mock-Vac mice (NVNC and Mock-Vac lines overlap). Theprime-boost vaccination protocol significantly increased levels ofantibodies against B. pertussis in intranasally vaccinated mice tolevels comparable to those in convalescent mice.

Six months after the initial prime vaccination, mice in each groupexcept convalescent mice and NVNC mice were challenged by B. pertussisinfection. Three days post challenge, mice were euthanized, and theeffect of the vaccine was tested, e.g., by flow cytometry or serology.As shown in FIG. 18, six months after vaccination, intranasalvaccination produced significant B. pertussis and PT antigen specificIgG titers in serum. These antibodies were not seen in NVNC mice orMock-Vac mice. Convalescent mice did not show PT antigen specificantibodies, but did show B. pertussis antibodies.

As seen in FIG. 18, exposure to an aP vaccine alone, or to an aP vaccinein combination with aluminum hydroxide or curdlan, increases antibodiesto the PT antigen significantly (P<0.05, compared to mock-vaccinatedmice). Exposure to an aP vaccine in combination with 1,3-beta-glucan hasa greater impact on levels of antibodies to the PT antigen (P<0.01,compared to mock-vaccinated mice). Further, the increase in total B.pertussis antibody titers in convalescent mice is significant, whencompared to mock-vaccinated mice (P<0.01). As seen in FIG. 18, theincrease in total B. pertussis antibody titers in mice treated with aP,aP-alum, or aP-curdlan vaccines is significant; however, it is lesssignificant that the increase in B. pertussis antibody titers inconvalescent mice (P<0.05 for the aP vaccine; P<0.01 for theconvalescent mice). The increase in total B. pertussis antibody titersin mice vaccinated with an aP vaccine in combination with1,3-beta-glucan is highly significant, when compared to mock-vaccinatedmice (P<0.001). Based on these results, it appears that administrationof an aP vaccine in combination with 1,3-beta-glucan increases antibodyproduction more than administration of an aP vaccine alone or withaluminum hydroxide or curdlan.

Example 8: Antibody-Expressing Cells in the Bone Marrow of VaccinatedMice

Antibody secreting cells (ASCs) are differentiated cells of the humoralimmune response. ASCs differentiate from activated B cells in lymphnodes. Most of the circulating ASCs undergo apoptosis, but some ASCsmigrate to the bone marrow (BM) and eventually mature into long-livedplasma cells (LLPCs). Accordingly, the bone marrow of mice vaccinated asin Example 6 was examined following euthanasia for the presence of ASCswhich secrete B. pertussis antibodies.

An enzyme-linked immune absorbent spot (ELISpot) analysis was used fordetecting antibody-secreting cells in bone marrow tissue, in response toB. pertussis infection. Data were recorded 3 days (FIG. 19A) and 7 days(FIG. 19B) post-challenge, as number of IgG spots per 3×10⁵ cells.Intranasal vaccination with aP, aP-alum, and aP-β-glucan (shown asaP+IRI-1501) significantly increased the number of B. pertussisantibody-secreting cells in bone marrow. Moreover, intranasalvaccination with aP-alum and aP-β-glucan significantly increased thenumber of B. pertussis antibody-secreting cells in bone marrow by threedays post-challenge, when compared to vaccination with the aP vaccinealone (FIG. 19A). By seven days post-challenge,

Although the various embodiments have been described in detail withparticular reference to certain aspects thereof, it should be understoodthat the invention is capable of other embodiments and its details arecapable of modifications in various obvious respects. As is readilyapparent to those skilled in the art, variations and modifications canbe affected while remaining within the spirit and scope of theinvention. Accordingly, the foregoing disclosure, description, andfigures are for illustrative purposes only and do not in any way limitthe invention, which is defined only by the claims.

1. A vaccine composition, comprising: a Bordetella pertussis antigen,and an effective adjuvant amount of a high molecular weight glucosepolymer.
 2. The vaccine composition of claim 1, wherein the highmolecular weight glucose polymer has a molecular weight of between 68kDal and 680 kDal.
 3. The vaccine composition of claim 1, wherein thehigh molecular weight glucose polymer is soluble or dispersible in wateror aqueous base, and gellable in the presence in the presence of CO₂ andaqueous acid.
 4. The vaccine composition of claim 1, wherein the highmolecular weight glucose polymer is a beta-glucan.
 5. The vaccinecomposition of claim 1, wherein the high molecular weight glucosepolymer is a 1,3-beta-glucan polymer.
 6. The vaccine composition ofclaim 1, wherein the high molecular weight glucose polymer is a1,3-beta-glucan polymer, a 1,3-beta-glucan/1,4-beta-glucan copolymer, a1,3-beta-glucan/1,6-beta-glucan copolymer, or a mixture thereof.
 7. Thevaccine composition of claim 4, wherein the β-glucan is selected fromthe group consisting of curdlan, dextran, and a1,3-beta-glucan/1,6-beta-glucan copolymer derived from baker's yeast. 8.The vaccine composition of claim 1, wherein the Bordetella pertussisantigen is selected from the group consisting of an extracellular toxin,an adhesion protein, an outer membrane protein, a receptor protein,fragments thereof, and mixtures thereof.
 9. The vaccine composition ofclaim 1, wherein the Bordetella pertussis antigen is selected from thegroup consisting of an extracellular pertussis toxin (PT), the adhesionproteins filamentous hemagglutinin (FHA) and fimbriae (FIM)), the outermembrane protein pertactin (PRN), the siderophore receptor protein FauA,the xenosiderophore receptor protein BfeA, the hemophore receptorprotein BfuR, fragments thereof, and mixtures thereof.
 10. The vaccinecomposition of claim 9, wherein the Bordetella pertussis antigen isselected from the group consisting of the extracellular pertussis toxin(PT), the adhesion protein filamentous hemagglutinin (FHA), thesiderophore receptor protein FauA, fragments thereof, and mixturesthereof.
 11. The vaccine composition of claim 1, wherein the compositionfurther includes an adenylate cyclase toxin (ACT) antigen.
 12. Thevaccine composition of claim 11, wherein the ACT antigen is a C-terminalrepeats-in-toxin domain (RTX) of ACT.
 13. The vaccine composition ofclaim 1, wherein the composition is formulated to induce a Th1/Th17immune response.
 14. The vaccine composition of claim 1, wherein thecomposition is formulated for intranasal administration.
 15. The vaccinecomposition of claim 1, wherein the composition is formulated forparenteral administration by subcutaneous (SC) injection, transdermaladministration, intramuscular (IM) injection, or intradermal (ID)injection.
 16. The vaccine composition of claim 1, wherein thecomposition is formulated for non-parenteral administration by oraladministration, intravaginal administration, pulmonary administration,ophthalmic administration, or rectal administration.
 17. A vaccinecomposition for intranasal administration to a patient, comprising: aBordetella pertussis antigen, and an effective adjuvant amount of a highmolecular weight glucose polymer, wherein the high molecular weightglucose polymer is configured to adhere to the airway of a patient, byforming a gel in the presence in the presence of CO₂ and aqueous acid.18. A method of immunizing a host against pertussis by administering thecomposition of claim 1 intranasally to the host.
 19. A method ofenhancing the immune response of an intranasally administered B.pertussis antigen that involves co-administering the antigen and a highmolecular weight glucose polymer.
 20. A vaccine composition, comprising:a Bordetella pertussis antigen, and an effective amount of a largeparticle adjuvant.
 21. The vaccine composition of claim 20, wherein thelarge particle comprises a high molecular weight glucose polymer. 22.The vaccine composition of claim 21, wherein the high molecular weightglucose polymer has a molecular weight of between 68 kDal and 680 kDal.23. The vaccine composition of claim 20, wherein the large particle hasa minimum dimension of about 3 μm.
 24. The vaccine composition of claim23, wherein the large particle comprises an aluminum hydroxide particle.25. The vaccine composition of claim 23, wherein the large particlecomprises a high molecular weight glucose polymer.
 26. The vaccinecomposition of claim 25, wherein the high molecular weight glucosepolymer is a 1,3-beta-glucan polymer, a 1,3-beta-glucan/1,4-beta-glucancopolymer, a 1,3-beta-glucan/1,6-beta-glucan copolymer, or a mixturethereof.
 27. The vaccine composition of claim 20, wherein the Bordetellapertussis antigen is selected from the group consisting of anextracellular pertussis toxin (PT), the adhesion proteins filamentoushemagglutinin (FHA) and fimbriae (FIM)), the outer membrane proteinpertactin (PRN), the siderophore receptor protein FauA, thexenosiderophore receptor protein BfeA, the hemophore receptor proteinBfuR, fragments thereof, and mixtures thereof.
 28. The vaccinecomposition of claim 20, wherein the composition further includes anadenylate cyclase toxin (ACT) antigen.
 29. The vaccine composition ofclaim 28, wherein the ACT antigen is a C-terminal repeats-in-toxindomain (RTX) of ACT.
 30. The vaccine composition of claim 20, whereinthe composition is formulated for intranasal administration.
 31. Thevaccine composition of claim 20, wherein the composition is formulatedfor parenteral administration by subcutaneous (SC) injection,transdermal administration, intramuscular (IM) injection, or intradermal(ID) injection.
 32. The vaccine composition of claim 20, wherein thecomposition is formulated for non-parenteral administration by oraladministration, intravaginal administration, pulmonary administration,ophthalmic administration, or rectal administration.